Stable compositions for measuring human natriuretic peptides

ABSTRACT

The present invention relates to methods for making stable compositions, such as, but not limited to, test samples, that can be used in ligand-binding assays.

RELATED APPLICATION INFORMATION

This application is a continuation-in-part of U.S. application Ser. No.10/620,475, filed on Jul. 16, 2003, which is hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to stable compositions, including, but notlimited to, calibrators, controls and test samples, that can be used inligand-binding assays, such as immunoassays, and methods for making saidcompositions.

BACKGROUND OF THE INVENTION

Atrial natriuretic peptide (hereinafter referred to as “ANP”), brainnatriuretic peptide (hereinafter referred to as “BNP”), C-typenatriuretic peptide (hereinafter referred to as “CNP”) and Dendroaspisnatriuretic peptide (hereinafter referred to as “DNP”) are each membersof a family of hormones known as “natriuretic peptides”. ANP and BNPshare a wide spectrum of biological properties and belong to the cardiacnatriuretic system. Both ANP and BNP are of myocardial cell origin whileCNP is of endothelial cell origin. DNP was isolated from the venom ofthe green mamba snake and possesses structural similarity to ANP, BNPand CNP.

BNP received its name because it was first isolated from porcine brain,thus “BNP” stood for “brain natriuretic peptide”. However, because BNPbelongs to the cardiac natriuretic system, “brain” has been changed to“B-type”. Therefore, “BNP” now refers to “B-type natriuretic peptide”.

ANP is secreted by the heart in the atria. BNP is secreted by the heartthrough the coronary sinus, predominantly from the cardiac ventricles.BNP is secreted as a 108 amino acid polypeptide precursor (See Valli etal., J. Lab. Clin. Med., 134(5):437-444 (November 1999)). The matureform of BNP is made up of 32 amino acids with a 17 amino acid ringclosed by a disulfide bond between two cysteine residues, anamino-terminal tail of 9 amino acids, and a carboxyl-terminal tail of 6amino acids. ANP and CNP also have a 17 amino acid ring closed by adisulfide bond between two cysteine residues. Eleven of the seventeenamino acids in the ring are conserved between the three molecules. Inaddition to the 17 amino acid ring structure, ANP has an amino-terminaltail of 6 amino acids and a carboxy-terminal tail of 5 amino acids. ANPis produced as a 126 amino acid pro-ANP form that is the major storageform of ANP. After proteolytic cleavage between amino acids 98 and 99,the mature 28 amino acid peptide ANP is found in coronary sinus plasma(See Yandle, J. Internal Med., 235:561-576 (1994)).

CNP is found in the brain and cerebral spinal fluid and is the mostprevalent of the three peptides in the central nervous system. Little ifany CNP is present in the heart. Pro-CNP is a 103 amino acid peptidethat is processed into either CNP-53 (amino acids 51 to 103) or CNP-22(amino acids 82 to 103) that are the active peptides. In addition the 17amino acid ring structure, CNP-22 has an amino-terminal tail of 5 aminoacids and contains no carboxy-terminal tail. CNP-53 is identical toCNP-22 except for a 31 amino acid extension at the amino terminal end.

As mentioned previously, DNP was isolated from the venom of the greenmamba snake. The mature form of DNP is made up of 38 amino acids.DNP-like immunoreactivity (DNP-LI) has been reported in human plasma andthe plasma concentration of DNP-LI has been found to be elevated inpatients with congestive heart failure (See, Cataliotti, et al., MayoClin. Proc., 76:111-1119 (2001)). Additionally, it is also known thatthe infusion of synthetic DNP results in marked natriuresis and diuresisin association with increased plasma and urinary cyclic guanosinemonophosphate. Id.

The measurement of BNP in human plasma in the general population hasbeen found to reflect cardiac diseases, such as congestive heartfailure, ischemic heart diseases, atrial fibrillation and renaldysfunction. In fact, elevated levels of BNP in human plasma has beenreported in heart disease, following acute myocardial infarction andduring symptomless or subclinical ventricular dysfunction (See Mukoyamaet al., J. Clin. Invest., 87:11402-11412 (1991), Motwani et al., Lancet,341:1109-1113 (1993), Yoshibayashi et al., New Eng. J. Med., 327:434(1992)). Increased circulating levels of ANP are seen in congestiveheart failure, chronic renal failure and in severe hypertension. Thepresence of CNP in human plasma remains controversial with reports ofits absence or presence as CNP-22 (See Yandle, J. Internal Med.,235:561-576 (1994)).

A ligand binding assay is an analytical technique for measuringconcentrations of substances commonly referred to as ligands that reactselectively with specific binding proteins. Immunoassays that measurethe concentrations of antigens that react selectively with specificantibodies are an example of a class of ligand binding assays.

Ligand binding assays, such as immunoassays, for measuring humannatriuretic peptides in plasma are well-known in the art and arecommercially available. These immunoassays require the use of at leastone or two specific antibodies as well as at least one calibrator and,ideally, at least one control. Calibrators are used in ligand bindingassays to calibrate instruments prior to calculating the sample result.The calibrators and controls used in such assays are typically made fromhuman synthetic natriuretic peptides. Human synthetic natriureticpeptides are commercially available from a variety of sources. Forexample, human synthetic BNP is commercially available from PeptideInstitute (Osaka, Japan), American Peptide Company, Inc. (Sunnyvale,Calif.), Synpep Corporation (Dublin, Calif.) and PhoenixPharmaceuticals, Inc. (Belmont, Calif.).

One of the problems with both natural and synthetic human natriureticpeptides is that they are unstable in plasma and serum. Specifically,enzymes, such as proteases, cleave these peptides. For example,proteases cleave BNP (natural and synthetic) at various locations alongits amino acid chain. For example, protease cleavage is known to occurat the amino terminus of BNP between amino acids 2-3 (Shimizu et al.,Clinica Chimica Acta, 316:129-135 (2002)) and at its carboxy terminusbetween amino acids 30-32. Moreover, endopeptidase cleavage of BNP isalso known in the art (Davidson and Struthers, J. Hypertension,12:329-336 (1994)). Such cleavage is problematic because in order forcalibrators and controls to function properly in an assay, a humannatriuretic peptide containing immunoreactive or specific ligand bindingsites must be present at the intended concentration during the assay.

Thereupon, as a result of this instability, the calibrators and controlsused in such assays are sold either in lyophilized form (such as in theShionoria assay available from Shionogi & Co., Ltd., Osaka, Japan) orfrozen (such as in the Triage® assay available from Biosite, Inc., SanDiego, Calif.). Calibrators and controls in lyophilized form must bereconstituted in a solvent prior to use in an assay. Once reconstituted,these calibrators and controls must be used within a specific timebecause they are very unstable. Calibrators and controls that are frozen(usually at temperatures of about −20° C. or colder) remain frozen untilthawed for use in the assay. These calibrators and controls are thawedat room temperature and then vortexed or inverted to make thecalibrators and controls homogenous prior to testing. Frozen calibratorsand controls cannot be re-frozen and can only be used once (i.e. aresingle-use) and then discarded.

In addition to the calibrators and controls, immunoassays require theuse of at least one test sample. Test samples are normally biologicalsamples derived from serum, plasma, whole blood or other bodily fluids(normally from a human patient). The levels of at least one humannatriuretic peptide in the test sample is quantified in the immunoassay.Some samples, with extremely high levels of natriuretic peptides, mustbe diluted prior to analysis in order to get an accurate quantificationof the peptide level. However, as described earlier, one of the problemswith natural human natriuretic peptides, even in test samples, is thatthey are unstable. Thereupon, as a result of this instability, after atest sample is obtained and once it is diluted, it must be used in animmunoassay within a short period of time (i.e., less than one hour, seeADVIA Centaur® Assay Manual 06300497, Rev. C, 2003-06). Such a shortperiod of instability can be problematic for a laboratory conductingsuch the immunoassays, particularly if the volume of immunoassays to beconducted is large.

Thereupon, there is a need in the art for new calibrators and controlsfor use in human natriuretic ligand binding assays that are stable forextended periods of time and are easy and quick to use and do not needto be reconstituted or thawed prior to use in such assays. Additionally,there is also a need in the art for test samples that can be used insuch assays that are stable for extended periods of time.

SUMMARY OF THE INVENTION

In one embodiment, the present invention relates to stable liquidcalibrators and controls that can be used in ligand binding assays, suchas immunoassays, for measuring the level of natriuretic peptides in atest sample. The stable liquid calibrators and controls of the presentinvention have a pH of from about 4.0 to about 6.5, preferably fromabout 5.0 to about 6.0.

Moreover, the calibrators and controls of the present invention compriseat least one natriuretic peptide, preferably at least one humansynthetic natriuretic peptide, such as a human synthetic atrialnatriuretic peptide, human synthetic B-type natriuretic peptide, humansynthetic C-type natriuretic peptide or human synthetic Dendroaspisnatriuretic peptide. In addition to the natriuretic peptide, thecalibrators and controls can also comprise at least one buffer, or atleast one acid, or at least one base, or combinations of at least onebuffer, at least one acid and/or at least one base. Examples of buffersthat can be used include, but are not limited to, acetate buffers (suchas sodium acetate/acetic acid, potassium acetate/acetic acid), citratebuffers (such as sodium citrate/citric acid, potassium citrate/citricacid), phosphate buffers (such as mono-/di-sodium phosphate) orcombinations thereof. Examples of acids that can be used include, butare not limited to, acetic acid, citric acid,diethylenetriaminepentaacetic acid, hydrochloric acid or combinationsthereof. Examples of bases that can be used include, but are not limitedto, sodium hydroxide.

The calibrators and controls can also comprise at least one diluent. Thediluent can comprise at least one natriuretic stabilizing compound andat least one biocide. Examples of natriuretic stabilizing compounds thatcan be used include, but are not limited to, at least one protein orpolymer. Examples of proteins that can be used include, but are notlimited to, bovine serum albumin, bovine gamma globulin, or non-fat drymilk. Examples of polymers that can be used include, but are not limitedto, polyethylene glycol, dextran, dextran sulfate or polyvinylpyrrolidone. The diluent can further contain at least one buffer, atleast one acid, at least one base, or combinations thereof. Examples ofbuffers that can be used include, but are not limited to, acetatebuffers (such as sodium acetate/acetic acid, potassium acetate/aceticacid), citrate buffers (such as sodium citrate/citric acid, potassiumcitrate/citric acid), phosphate buffers (such as mono-/di-sodiumphosphate) or combinations thereof. Examples of acids that can be usedinclude, but are not limited to, acetic acid, citric acid,diethylenetriaminepentaacetic acid, hydrochloric acid or combinationsthereof. Examples of bases that can be used include, but are not limitedto, sodium hydroxide.

The calibrators and controls of the present invention remain stable forlong periods of time under a variety of storage conditions.Specifically, the calibrators and controls of the present inventionremain stable when stored for extended periods of time at a temperatureof from about 2 to about 8° C. Additionally, the calibrators andcontrols of the present invention remain stable when used at ambienttemperatures and for limited periods of time (such as between 1 minuteand 60 minutes, preferably for a period of about 20 to about 30 minutes)at temperatures of from about 30 to about 40° C., preferably at atemperature of about 34° C.

In a second embodiment, the present invention relates to a method ofmaking stable liquid calibrators and controls for use in a ligandbinding assay. The first step of the method involves mixing at least onediluent with at least one human synthetic natriuretic peptide to form aliquid calibrator or control. The second step involves measuring the pHof the liquid calibrator or control. Depending upon the pH of the liquidcalibrator or control measured in the first step, the pH of the liquidcalibrator or control may be adjusted so that the pH is in the range offrom about 4.0 to about 6.5, preferably in the range from about 5.0 toabout 6.0. The pH of the calibrator or control can be adjusted using atleast one buffer, or at least one acid, or at least one base orcombinations of at least one buffer, at least one acid and/or at leastone base. Examples of buffers that can be used include, but are notlimited to, acetate buffers (such as sodium acetate/acetic acid,potassium acetate/acetic acid), citrate buffers (such as sodiumcitrate/citric acid, potassium citrate/citric acid), phosphate buffers(such as mono-/di-sodium phosphate) or combinations thereof. Examples ofacids that can be used include, but are not limited to, acetic acid,citric acid, diethylenetriaminepentaacetic acid, hydrochloric acid orcombinations thereof. An example of a base that can be used is sodiumhydroxide.

The human synthetic natriuretic peptide used in the above-describedmethod can be human synthetic atrial natriuretic peptide, humansynthetic B-type natriuretic peptide, human synthetic C-type natriureticpeptide or human synthetic Dendroaspis natriuretic peptide.

The diluent used in above described method can comprise at least onenatriuretic stabilizing compound and at least one biocide. Examples ofnatriuretic stabilizing compounds, include, but are not limited to, atleast one protein or polymer. Examples of proteins that can be usedinclude, but are not limited to, bovine serum albumin, bovine gammaglobulin or non-fat dry milk. Examples of polymers that can be usedinclude, but are not limited to, polyethylene glycol, dextran, dextransulfate or polyvinyl pyrrolidone. The diluent can further contain atleast one buffer, at least one acid, at least one base, or combinationsthereof. Examples of buffers that can be used include, but are notlimited to, acetate buffers (such as sodium acetate/acetic acid,potassium acetate/acetic acid), citrate buffers (such as sodiumcitrate/citric acid, potassium citrate/citric acid), phosphate buffers(such as mono-/di-sodium phosphate) or combinations thereof. Examples ofacids that can be used include, but are not limited to, acetic acid,citric acid, diethylenetriaminepentaacetic acid, hydrochloric acid orcombinations thereof. Examples of bases that can be used include, butare not limited to, sodium hydroxide.

In a third embodiment, the present invention relates to a stable testsample that can be used in ligand binding assays, such as immunoassays,for measuring the level of at least one natriuretic peptide in said testsample. The stable test sample of the present invention has a pH of fromabout 4.0 to about 6.5, preferably from about 5.0 to about 6.0.

The test sample of the present invention comprises at least onebiological sample that contains at least one natriuretic peptide,preferably, at least one human natural natriuretic peptide, such as ahuman natural atrial natriuretic peptide, human natural B-typenatriuretic peptide, human natural C-type natriuretic peptide or humannatural Dendroaspis natriuretic peptide. The biological sample used inthe test sample is derived from human serum, human plasma, human wholeblood or other human bodily fluids. The test sample can also comprise atleast one diluent. The amount of diluent in the test sample is fromabout 5% to about 95% (v/v), preferably from about 20% to about 90%(v/v). The diluent can comprise at least one natriuretic stabilizingcompound and, optionally, at least one biocide. Examples of natriureticstabilizing compounds that can be used include, but are not limited to,at least one protein or polymer. Examples of proteins that can be usedinclude, but are not limited to, bovine serum albumin, bovine gammaglobulin, or non-fat dry milk. Examples of polymers that can be usedinclude, but are not limited to, polyethylene glycol, dextran, dextransulfate or polyvinyl pyrrolidone. The diluent can further contain atleast one buffer, or at least one acid, or at least one base, orcombinations of at least one buffer, at least one acid and/or at leastone base. Examples of buffers that can be used include, but are notlimited to, acetate buffers (such as sodium acetate/acetic acid,potassium acetate/acetic acid), citrate buffers (such as sodiumcitrate/citric acid, potassium citrate/citric acid), phosphate buffers(such as mono-/di-sodium phosphate) or combinations thereof. Examples ofacids that can be used include, but are not limited to, acetic acid,citric acid, diethylenetriaminepentaacetic acid, hydrochloric acid orcombinations thereof. Examples of bases that can be used include, butare not limited to, sodium hydroxide.

The test sample of the present invention remains stable for long periodsof time under a variety of storage conditions. Specifically, the testsample of the present invention remain stable when used at ambienttemperatures (for a period of at least about twenty-four (24) hours).

In a fourth embodiment, the present invention relates to a method ofmaking a stable test sample for use in a ligand binding assay. Themethod involves mixing from about 5% to about 95% (v/v), preferably fromabout 20% to about 90% (v/v), of at least one diluent with at least onea biological sample derived from serum, plasma, whole blood or otherbodily fluids, to form a stable test sample having a pH of from about4.0 to about 6.5, preferably in the range from about 5.0 to about 6.0.The biological sample contains at least one natriuretic peptide that canbe a human natriuretic peptide, such as, but not limited to, a humannatural atrial natriuretic peptide, human natural B-type natriureticpeptide, human natural C-type natriuretic peptide or human naturalDendroaspis natriuretic peptide. The biological sample is derived fromserum, plasma, whole blood or other bodily fluids.

The diluent used in above described method can comprise at least onenatriuretic stabilizing compound and, optionally, at least one biocide.Examples of natriuretic stabilizing compounds, include, but are notlimited to, at least one protein or polymer. Examples of proteins thatcan be used include, but are not limited to, bovine serum albumin,bovine gamma globulin or non-fat dry milk. Examples of polymers that canbe used include, but are not limited to, polyethylene glycol, dextran,dextran sulfate or polyvinyl pyrrolidone. The diluent can furthercontain at least one buffer, or at least one acid, or at least one base,or combinations of at least one buffer, at least one acid and/or atleast one base . Examples of buffers that can be used include, but arenot limited to, acetate buffers (such as sodium acetate/acetic acid,potassium acetate/acetic acid), citrate buffers (such as sodiumcitrate/citric acid, potassium citrate/citric acid), phosphate buffers(such as mono-/di-sodium phosphate) or combinations thereof. Examples ofacids that can be used include, but are not limited to, acetic acid,citric acid, diethylenetriaminepentaacetic acid, hydrochloric acid orcombinations thereof. Examples of bases that can be used include, butare not limited to, sodium hydroxide.

In a fifth embodiment, the present invention relates to a method ofstabilizing a test sample for use in a ligand binding assay. The methodinvolves mixing from about 5% to about 95% (v/v), preferably from about20% to about 90% (v/v), of at least one diluent with at least one abiological sample derived from serum, plasma, whole blood or otherbodily fluids, to form a stable test sample having a pH of from about4.0 to about 6.5, preferably in the range from about 5.0 to about 6.0.The biological sample contains at least one natriuretic peptide that canbe a human natriuretic peptide, such as, but not limited to, a humannatural atrial natriuretic peptide, human natural B-type natriureticpeptide, human natural C-type natriuretic peptide or human naturalDendroaspis natriuretic peptide. The biological sample is derived fromserum, plasma, whole blood or other bodily fluids.

The diluent used in above described method can comprise at least onenatriuretic stabilizing compound and, optionally, at least one biocide.Examples of natriuretic stabilizing compounds, include, but are notlimited to, at least one protein or polymer. Examples of proteins thatcan be used include, but are not limited to, bovine serum albumin,bovine gamma globulin or non-fat dry milk. Examples of polymers that canbe used include, but are not limited to, polyethylene glycol, dextran,dextran sulfate or polyvinyl pyrrolidone. The diluent can furthercontain at least one buffer, or at least one acid, or at least one base,or combinations of at least one buffer, at least one acid and/or atleast one base . Examples of buffers that can be used include, but arenot limited to, acetate buffers (such as sodium acetate/acetic acid,potassium acetate/acetic acid), citrate buffers (such as sodiumcitrate/citric acid, potassium citrate/citric acid), phosphate buffers(such as mono-/di-sodium phosphate) or combinations thereof. Examples ofacids that can be used include, but are not limited to, acetic acid,citric acid, diethylenetriaminepentaacetic acid, hydrochloric acid orcombinations thereof. Examples of bases that can be used include, butare not limited to, sodium hydroxide.

The stabilized test sample prepared by the above-identified method canbe used immediately after preparation in a ligand binding assay, or canbe used at a much later period in time in a ligand binding assay. Forexample, when the stabilized test sample is maintained in a laboratoryconditions at ambient temperatures, it can be used in a ligand bindingassay at least twenty-four (24) hours after being stabilized pursuant tothe above-described method.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1P show graphs demonstrating the stability of calibratorshaving varying pH's at 37° C. over time.

FIGS. 2A-2P show graphs demonstrating the stability of calibratorshaving varying pH's at 2-8° C. over time.

FIGS. 3A-3T show graphs demonstrating the stability of calibratorshaving varying pH's at 37° C. and 2-8° C. over time.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention relates to stable calibratorsand controls that can be used in ligand binding assays, such asimmunoassays. More specifically, the present invention relates to stablecalibrators and controls that are in liquid form and can be used inligand binding assays to measure the levels of at least one natriureticpeptide in a test sample. The test sample comprises at least one abiological sample that is derived from plasma, serum, whole blood orother bodily fluids, such as, but not limited to, saliva, sputum, etc.,as long as the bodily fluids are stable in their collection technique.The biological sample also contains at least one natriuretic peptide.Because the calibrators and controls of the present invention are inliquid form, they do not need to be reconstituted or defrosted prior touse like the lyophilized and frozen calibrators and controls known inthe art. Additionally, the calibrators and controls of the presentinvention are stable for long periods of time under a variety of storageconditions and temperatures.

The calibrators and controls of the present invention contain at leastone natriuretic peptide and thus can be used in ligand binding assays tomeasure the level of these peptides in a test sample. Either natural orsynthetic natriuretic peptides can be used in the calibrators andcontrols of the present invention. Preferably, the natriuretic peptidesare synthetic natriuretic peptides and are human, such as, humansynthetic atrial natriuretic peptide, human synthetic B-type natriureticpeptide, human synthetic C-type natriuretic peptide or human syntheticDendroaspis natriuretic peptide. Human synthetic atrial natriureticpeptides, human synthetic B-type natriuretic peptides, human syntheticC-type natriuretic peptides and human synthetic Dendroaspis natriureticpeptides are commercially available from a variety of sources,including, but not limited to, Peptide Institute (Osaka, Japan),American Peptide Company, Inc. (Sunnyvale, Calif.), Synpep Corporation(Dublin, Calif.) and Phoenix Pharmaceuticals, Inc. (Belmont, Calif.).The amount of natriuretic peptides in the calibrators of the presentinvention is from 0 to about 10,000 pg/mL, preferably in the amount offrom about 25 to about 5,000 pg/mL. The amount of natriuretic peptidesin the controls of the present invention is from about 25 to about 4000pg/mL, preferably in the amount of from about 40 to about 2000 pg/mL.

In addition to the natriuretic peptides, the calibrators and controlsare in a diluent that comprises a number of components. Any diluent canbe used in the calibrators and controls of the present invention. Forexample, a diluent that can be used in the calibrators and controls ofthe present invention can be made using techniques known in the art orcan be a purchased from a commercially available source. Combinations ofboth custom made diluents and commercially purchased diluents are alsocontemplated within the scope of the present invention.

The composition of the diluent can vary depending upon the calibrator orcontrol. For example, the diluent can comprise water.

The diluent can also comprise at least one natriuretic stabilizingcompound. As used herein, the term “natriuretic stabilizing compound”refers to a compound that can be used to stabilize a natriuretic peptideand prevent its degradation by enzymes, such as proteases. Severalnatriuretic stabilizing compounds are known in the art and can be usedin the calibrators and controls of the present invention. Thesecompounds include, but are not limited to, proteins, polymers andprotease inhibitors. Examples of proteins that can be used include, butare not limited to, bovine serum albumin (BSA), bovine gamma globulin(bovine IgG), or non-fat dry milk (such as Nestlé® Carnation® Nonfat DryMilk). Examples of polymers that can be used include, polyethyleneglycol (having a molecular weight of from about 2,000 to about 20,000daltons), dextran (having a molecular weight of from about 5,000 toabout 670,000 daltons), dextran sulfate (having a molecular weight ofabout 5,000 daltons or about 1,000,000 daltons) or polyvinyl pyrrolidone(having a molecular weight of about 40,000 daltons). Bovine serumalbumin, bovine IgG, polyethylene glycol, dextran, dextran sulfate andpolyvinyl pyrrolidone can all be purchased from Sigma Aldrich, St.Louis, Mo. Examples of protease inhibitors that can be used include, butare not limited to, EDTA or aprotinin. It is known in the art that EDTAat about lmg/mL blood and aprotinin at approximately 50 kIU/mL of blood(Murdoch et al, Heart, 78:594-597 (1997)) can be used to help preventBNP proteolysis. Protease inhibitors, such as EDTA and aprotinin can bepurchased from Sigma Aldrich, St. Louis, Mo. The amount of natriureticstabilizing compound in the diluent can be from about 0.01 mg/mL toabout 400 mg/mL, preferably in the amount of from about 1 mg/mL to about50 mg/mL.

Additionally, the diluent can also comprise and at least one biocideand/or preservative. As used herein, the term “biocide” refers to asubstance that can be used to kill a variety of different organisms.Suitable biocides and/or preservatives for use in the diluent can bedetermined using routine techniques by those skilled in the art.Examples of suitable biocides and/or preservatives that can be used inthe present invention, include, but are not limited to ProClin® 300(Sigma-Aldrich, St. Louis, Mo.) (The active ingredients of ProClin® 300are 5-chloro-2-methyl-4-isothiazolin-3-one and2-methyl-4-isothiazolin-3-one) and sodium azide. ProClin® 300 alsocontains a number of inert ingredients such as a modified glycol andalkyl carboxylate. The amount of biocide and/or preservative in thediluent can be from about 0.001 mg/mL to about 50 mg/mL, preferably inthe amount of from about 0.1 mg/mL to about 10 mg/mL.

Furthermore, the diluent can also comprise at least one buffer, or atleast one acid, or at least one base or combinations of at least onebuffer, at least one acid and/or at least one base . Examples of buffersthat can be used include, but are not limited to, acetate buffers (suchas sodium acetate/acetic acid, potassium acetate/acetic acid), citratebuffers (such as sodium citrate/citric acid, potassium citrate/citricacid), phosphate buffers (such as mono-/di-sodium phosphate) orcombinations thereof. It is preferred that the buffer have a goodbuffering capacity at the desired pH so that it can stabilize the pH ofthe diluent and the resulting calibrator or control. Examples of acidsthat can be used include, but are not limited to, acetic acid,diethylenetriaminepentaacetic acid (DTPA), hydrochloric acid (HCI) orcombinations thereof. The amount of buffer or acid in the diluent can befrom about 1 mM to about 500 mM, preferably in the amount of from about5 to about 200 mM. An example of a base that can be used includes, butis not limited to, sodium hydroxide (NaOH). The amount of base in thediluent can be from about 0.01 mM to about 50 mM, preferably in theamount of from about 1.0 mM to about 10 mM.

The diluent can also comprise sodium chloride (NaCl) and/or at least onedetergent, such as Tween® (Any type of Tween® can be used, including,but not limited to, Tween® 20, Tween® 40, Tween® 60, which arecommercially available from Sigma-Aldrich, St. Louis, Mo.). The amountof NaCl in the diluent can be from about 25 mM to about 500 mM,preferably in the amount of from about 100 mM to about 400 mM. Theamount of detergent in the diluent can be from about 0.01 mg/mL to about10 mg/mL, preferably in the amount of from about 0.1 mg/mL to about 3.0mg/mL.

In addition, as mentioned above, commercially available diluents can beused. For example, the diluent known as “MEIA2” that is commerciallyavailable as IMx MEIA #2 Diluent Buffer (No.8374-04) from AbbottLaboratories, the assignee of the present invention, can be used. MEIA2contains 0.06 M Tris buffer (Sigma-Aldrich, St. Louis, Mo.), 0.3 M NaCl,0.1% w/v NaN₃ and has a pH of 7.5.

In addition to the diluent, the calibrators and controls can alsocontain at least one buffer, or at least one acid, or at least one base,or combinations of at least one buffer, at least one acid and/or atleast one base. These buffers, bases or acids can be present in thecalibrators and controls in addition to the buffers, bases and/or acidspresent in the diluent or as separate components if the diluent does notcontain any buffers, bases and/or acids. Examples of buffers that can beused include, but are not limited to, acetate buffers (such as sodiumacetate/acetic acid, potassium acetate/acetic acid), citrate buffers(such as sodium citrate/citric acid, potassium citrate/citric acid),phosphate buffers (such as mono-/di-sodium phosphate) or combinationsthereof. Examples of acids that can be used include, but are not limitedto, acetic acid, citric acid, diethylenetriaminepentaacetic acid (DTPA),hydrochloric acid (HCl) or combinations thereof. Examples of bases thatcan be used include, but are not limited to, sodium hydroxide. Theamount of buffer, acid, base or combinations thereof in the calibratoror control can be from about 1 mM to about 500 mM, preferably in theamount of from about 5 to about 200 mM.

As mentioned above, the calibrators and controls of the presentinvention are stable and are in liquid form. The key to the stability ofthe calibrators and controls of the present invention is pH. Morespecifically, it has been discovered that the liquid calibrators andcontrols of the present invention are stable at a pH of from about 4.0to about 6.5. Preferably, the pH of the liquid calibrators and controlsof the present invention is from about 5.0 to about 6.0.

The calibrators and controls of the present invention having a pH offrom about 4.0 to about 6.5 are stable for long periods of time andunder a variety of storage conditions. For example, the calibrators andcontrols of the present invention remain stable when stored attemperatures of from about 2 to about 8° C. for a period of about twelve(12) months or more. The ability to store the calibrators and controlsof the present invention at about 2° C. to about 8° C. is veryconvenient and allows for multiple-use of these reagents. In contrast,the frozen calibrators and controls known in the art are single-usereagents that have to be defrosted prior to conducting an assay. Thesecalibrators and controls cannot be refrozen and have to be thrown awayafter being defrosted and used in an assay. Lyophilized controls knownin the art, are only stable for 1 week at 2-8° C. after reconstitutionand must be discarded.

Additionally, the calibrators and controls of the present inventionhaving a pH of from about 4.0 to about 6.5 remain stable for severalhours when used under laboratory conditions at ambient temperatures.Additionally, the calibrators and controls of the present invention arestable for limited periods of time (such as, but not limited to, about 1minute to about 60 minutes, preferably for a period of about 20 minutesto about 30 minutes) when exposed to temperatures of from about 30 toabout 40° C., preferably at a temperature of about 34° C.

In another embodiment, the present invention relates to methods formaking stable calibrators and controls for use in ligand binding assays,such as immunoassays. The method of the present invention involvesmixing at least one diluent with at least one natriuretic peptide toform a liquid calibrator or control. The diluent can have any of thecompositions previously described herein and the natriuretic peptide canbe any of the natriuretic peptides also previously described herein.Preferably, the diluent comprises at least one buffer having a goodbuffering capacity at the desired pH. Optionally, once the diluent hasbeen prepared, it can optionally be heated to a temperature of fromabout 50 to about 60° C. for a period of from about 30 minutes to about24 hours. Preferably, the diluent is heated in a water bath at atemperature of about 55° C. for a period of about 1 hour.

The diluent and natriuretic peptide are mixed together at a temperatureof from about 15° C. to about 30° C., preferably at a temperature offrom about 19° C. to about 23° C., until a homogenous solution isobtained. Once a homogenous liquid calibrator or control has beenprepared, the pH is measured using a pH meter using techniques known inthe art. If the pH of the liquid calibrator or control is determined tobe in the range of from about 4.0 to about 6.5, then the pH of theliquid calibrator and control does not require any adjustment orstabilization and can be subjected to further processing steps such asthose described in more detail below. However, if the pH of the liquidcalibrator or control is not in the range of from about 4.0 to about6.5, then the pH is adjusted or stabilized using routine techniquesknown in the art. For example, if the pH of the liquid calibrator orcontrol is determined to be less than about 4.0, then at least one base,such as sodium hydroxide, at least one buffer, or combinations thereof,is added to the liquid calibrator or control in small quantities,preferably drop wise, and the pH measured repeatedly until the pH of theliquid calibrator or control is in the range of from about 4.0 to about6.5. Likewise, if the pH of the liquid calibrator or control isdetermined to be greater than 6.5, then at least one buffer, at leastone acid or combinations thereof is added to the liquid calibrator orcontrol in small quantities, preferably drop wise, and the pH measuredrepeatedly until the pH of the liquid calibrator or control is in therange of from about 4.0 to about 6.5. Examples of buffers that can beused include, but are not limited to, acetate buffers (such as sodiumacetate/acetic acid, potassium acetate/acetic acid), citrate buffers(such as sodium citrate/citric acid, potassium citrate/citric acid),phosphate buffers (such as mono-/di-sodium phosphate) or combinationsthereof. Examples of acids that can be used include, but are not limitedto, acetic acid, citric acid, diethylenetriaminepentaacetic acid,hydrochloric acid or combinations thereof.

Once a liquid calibrator or control having a pH in the range of fromabout 4.0 to about 6.5 has been prepared, then the calibrator or controlcan optionally be heated to a temperature of from about 50° to about 60°C. for a period of from about 30 minutes to about 24 hours. Preferably,the calibrator or control is heated in a water bath at a temperature ofabout 55° C. for a period of about 1 hour. After the calibrator orcontrol has been heated, it can then be subjected to further processingsteps such as those described below.

Once a liquid calibrator or control having a pH in the range of fromabout 4.0 to about 6.5 has been prepared, then the calibrator or controlcan be subjected to further processing steps, such as, but not limitedto, vortexing and/or filtering, using techniques known in the art. Oncethese steps have been completed, then the calibrator or control isplaced into bioclean or sterile vials (plastic or siliconized glass) andlabeled accordingly. Once the calibrators or controls are placed intothe bioclean or sterile vials, they can be stored for use in an assay ata temperature of from about 2° C. to about 8° C. for up to 12 months ormore.

In third embodiment, the present invention relates to stable testsamples that can be used in ligand binding assays, such as immunoassays.More specifically, the present invention relates to stable test samplesthat contain at least one biological sample that contains at least onenatriuretic peptide. The levels of the at least one natriuretic peptidein the biological sample can be quantified in the ligand binding assay.The biological sample is derived from plasma, serum, whole blood orother bodily fluids, such as, but not limited to, saliva, sputum, etc.,as long as the bodily fluids are stable in their collection technique.Additionally, the test samples of the present invention are stable forat least twenty-four (24) hours and can be used under a variety ofstorage conditions and temperatures.

As mentioned briefly above, the test sample contains at least onebiological sample that is derived from serum, plasma, whole blood orother bodily fluids and contains at least one naturiuretic peptide.Preferably, the naturiuretic peptide is a natural natriuretic peptidethat is human and obtained from or contained within a biological samplederived from human serum, human plasma, human whole blood or other humanbodily fluids. The human natriuretic peptide can be a human naturalatrial natriuretic peptide, human natural B-type natriuretic peptide,human natural C-type natriuretic peptide or human natural Dendroaspisnatriuretic peptide.

In addition to the biological sample(s), the test sample contains adiluent that comprises a number of components. Any diluent can be usedin the test sample of the present invention. For example, a diluent thatcan be used in the test sample of the present invention can be madeusing techniques known in the art or can be a purchased from acommercially available source. Combinations of both custom made diluentsand commercially purchased diluents are also contemplated within thescope of the present invention.

The composition of the diluent can vary depending upon the test sample.For example, the diluent can comprise water.

The diluent can also comprise at least one natriuretic stabilizingcompound. As used herein, the term “natriuretic stabilizing compound”refers to a compound that can be used to stabilize a natriuretic peptideand prevent its degradation by enzymes, such as proteases. Severalnatriuretic stabilizing compounds are known in the art and can be usedin the calibrators and controls of the present invention. Thesecompounds include, but are not limited to, proteins, polymers andprotease inhibitors. Examples of proteins that can be used include, butare not limited to, bovine serum albumin (BSA), bovine gamma globulin(bovine IgG), or non-fat dry milk (such as Nestlé® Carnation® Nonfat DryMilk). Examples of polymers that can be used include, polyethyleneglycol (having a molecular weight of from about 2,000 to about 20,000daltons), dextran (having a molecular weight of from about 5,000 toabout 670,000 daltons), dextran sulfate (having a molecular weight ofabout 5,000 daltons or about 1,000,000 daltons) or polyvinyl pyrrolidone(having a molecular weight of about 40,000 daltons). Bovine serumalbumin, bovine IgG, polyethylene glycol, dextran, dextran sulfate andpolyvinyl pyrrolidone can all be purchased from Sigma Aldrich, St.Louis, Mo. Examples of protease inhibitors that can be used include, butare not limited to, EDTA or aprotinin. It is known in the art that EDTAat about 1 mg/mL blood and aprotinin at approximately 50 kIU/mL of blood(Murdoch et al, Heart, 78:594-597 (1997)) can be used to help preventBNP proteolysis. Protease inhibitors, such as EDTA and aprotinin can bepurchased from Sigma Aldrich, St. Louis, Mo. The amount of natriureticstabilizing compound in the diluent can be from about 0.01 mg/mL toabout 400 mg/mL, preferably in the amount of from about 1 mg/mL to about50 mg/mL.

Additionally, the diluent can also comprise and at least one biocideand/or preservative. As used herein, the term “biocide” refers to asubstance that can be used to kill a variety of different organisms.Suitable biocides and/or preservatives for use in the diluent can bedetermined using routine techniques by those skilled in the art.Examples of suitable biocides and/or preservatives that can be used inthe present invention, include, but are not limited to, sodium azide andProClin® 300 (Sigma-Aldrich, St. Louis, Mo.) (The active ingredients ofProClin® 300 are 5-chloro-2-methyl-4-isothiazolin-3-one and2-methyl-4-isothiazolin-3-one. ProClin® 300 also contains a number ofinert ingredients such as a modified glycol and alkyl carboxylate). Theamount of biocide and/or preservative in the diluent can be from about0.001 mg/mL to about 50 mg/mL, preferably in the amount of from about0.1 mg/mL to about 10 mg/mL.

Furthermore, the diluent can also comprise at least one buffer, or atleast one acid, or at least one base or combinations of at least onebuffer, at least one acid and/or at least one base. Examples of buffersthat can be used include, but are not limited to, acetate buffers (suchas sodium acetate/acetic acid, potassium acetate/acetic acid), citratebuffers (such as sodium citrate/citric acid, potassium citrate/citricacid), phosphate buffers (such as mono-/di-sodium phosphate) orcombinations thereof. It is preferred that the buffer have a goodbuffering capacity at the desired pH so that it can stabilize the pH ofthe diluent and the resulting calibrator or control. Examples of acidsthat can be used include, but are not limited to, acetic acid,diethylenetriaminepentaacetic acid (DTPA), hydrochloric acid (HCl) orcombinations thereof. The amount of buffer or acid in the diluent can befrom about 1 mM to about 500 mM, preferably in the amount of from about5 to about 200 mM. An example of a base that can be used includes, butis not limited to, sodium hydroxide (NaOH). The amount of base in thediluent can be from about 0.01 mM to about 50 mM, preferably in theamount of from about 1.0 mM to about 10 mM.

The diluent can also comprise sodium chloride (NaCl) and/or at least onedetergent, such as Tween® (Any type of Tween® can be used, including,but not limited to, Tween® 20, Tween® 40, Tween® 60, which arecommercially available from Sigma-Aldrich, St. Louis, Mo.). The amountof NaCl in the diluent can be from about 25 mM to about 500 mM,preferably in the amount of from about 100 mM to about 400 mM. Theamount of detergent in the diluent can be from about 0.01 mg/mL to about10 mg/mL, preferably in the amount of from about 0.1 mg/mL to about 3.0mg/mL.

In addition, as mentioned above, commercially available diluents can beused. For example, the diluent known as “MEIA2” that is commerciallyavailable as IMx MEIA #2 Diluent Buffer (No.8374-04) from AbbottLaboratories, the assignee of the present invention, can be used afteradjustment of diluent buffer to desired pH as shown in Examples 1, 2 and3 below. MEIA2 contains 0.06 M Tris buffer (Sigma-Aldrich, St. Louis,Mo.), 0.3 M NaCl, 0.1% w/v NaN₃ and has a pH of 7.5.

The pH of the diluent used in the test sample is from about 4.0 to about6.0, preferably from about 5.4 to about 5.6. The amount of diluentcontained in the test sample is from about 5% to about 95% (v/v)preferably, from about 20% to about 90% (v/v).

As mentioned above, the test samples of the present invention arestable. The key to the stability of the test sample of the presentinvention is pH. More specifically, it has been discovered that the testsamples of the present invention are stable at a pH of from about 4.0 toabout 6.5. Preferably, the pH of the test samples of the presentinvention is from about 5.0 to about 6.0.

The test samples of the present invention having a pH of from about 4.0to about 6.5 are stable for at least twenty-four (24) hours when usedunder laboratory conditions at ambient temperatures. In contrast, testsamples known in the art are only stable for an hour or less after theyare diluted.

In another embodiment, the present invention relates to methods formaking stable test samples for use in ligand binding assays, such asimmunoassays. The method of the present invention involves mixing fromabout 5% to about 95% (v/v), preferably from about 20% to about 90%(v/v) of at least one diluent having a pH of from about 4.0 to about6.0, preferably from about 5.4 to about 5.6, with at least onebiological sample derived from plasma, serum, whole blood or otherbodily fluid and that contains at least one natriuretic peptide to forma stable, test sample. The diluent can have any of the compositionspreviously described herein and the natriuretic peptide can be any ofthe human natural natriuretic peptides also previously described herein.Preferably, the diluent comprises at least one buffer having a goodbuffering capacity at the desired pH.

The diluent and biological sample can be mixed together at a temperatureof from about 15° C. to about 30° C., preferably at a temperature offrom about 19° C. to about 23° C., until a homogenous solution isobtained. The pH of the resulting homogenous test sample can be in therange of from about 4.0 to about 6.5. After the stable test sample hasbeen prepared, it can be used in an immunoassay according to standardprotocols.

In another embodiment, the present invention relates to methods forstabilizing test samples for use in ligand binding assays, such asimmunoassays. The method of the present invention involves mixing fromabout 5% to about 95% (v/v), preferably from about 20% to about 90%(v/v) of at least one diluent having a pH of from about 4.0 to about6.0, preferably from about 5.4 to about 5.6, with at least onebiological sample derived from plasma, serum, whole blood or otherbodily fluid and that contains at least one natriuretic peptide to forma stable, test sample. The diluent can have any of the compositionspreviously described herein and the natriuretic peptide can be any ofthe human natural natriuretic peptides also previously described herein.Preferably, the diluent comprises at least one buffer having a goodbuffering capacity at the desired pH.

The diluent and biological sample can be mixed together at a temperatureof from about 15° C. to about 30° C., preferably at a temperature offrom about 19° C. to about 23° C., until a homogenous solution isobtained. The pH of the resulting homogenous test sample can be in therange of from about 4.0 to about 6.5. After the stabilized test samplehas been prepared, it can be used in an immunoassay according tostandard protocols. More specifically, the stabilized test can be usedimmediately after it has been prepared in a ligand binding assay, or canbe used at a much later period in time in a ligand binding assay. Forexample, when the stabilized test sample is maintained in a laboratoryconditions at ambient temperatures, it can be used in a ligand bindingassay at least about twenty-four (24) hours after being stabilizedpursuant to the above-described method.

By way of example, and not of limitation, examples of the presentinvention will now be given.

EXAMPLE 1 BNP Immunoassay

Bulk diluents (“Dil”) having the following formulations were prepared:

Experimental Control Diluent=4% BSA, 1% PEG, 0.1% Tween, 1% Dextran,0.1% Proclin® 300, MEIA2 diluent.

Dil1=2% BSA, 0.1% Proclin® 300, MEIA2 diluent

Dil2=Same as Dil1.

Dil3=2% BSA, 0.1% ProClin® 300, MEIA2 diluent and 0.1% Tween.

Dil4=Same as Dil3.

Dil5=2% BSA, 0.1% ProClin® 300, MEIA2 diluent and 10 mM DTPA

Dil6—Same as Dil5.

Dil7=2% BSA, 0.1% ProClin® 300, MEIA2 diluent, 0.1% Tween, 10 mM DTPA.

Dil8=Same as Dil7.

Dil9=Same as Dil1.

Dil10=Same as Dil2.

Dil11=Same as Dil3.

Dil12=Same as Dil4.

Dil13=Same as Dil5.

Dil14=Same as Dil6.

Dil15=Same as Dil7.

Dil16=Same as Dil8.

After each of the above diluents was prepared, the pH was determined.The pH of diluents 7-16 was adjusted. Specifically, for diluents thatdid not contain DTPA, HCl was added (drop wise) to adjust the pH (toabout 5.6). For the diluents that contained DTPA, NaOH was used toadjust the pH (to about 5.6 or 7.4). The pH was not adjusted fordiluents 1-4. The final pH for each diluent is shown below in Table 1.

TABLE 1 Dil1 - pH 7.41 Dil2 - pH 7.41* Dil3 - pH 7.39 Dil4 - pH 7.39*Dil5 - pH 7.41 Dil6 - pH 7.41* Dil7 - pH 7.40 Dil8 - pH 7.40* Dil9 - pH5.66 Dil10 - pH 5.66* Dil11 - pH 5.64 Dil12 - pH 5.64* Dil13 - pH 5.66Dil14 - pH 5.66* Dil15 - pH 5.64 Dil16 - pH 5.64* The pH of theexperimental control diluent was 7.4. *After final adjustment of the pH,these diluents (and the control) were also subjected to heat.Specifically, these diluents were heated in a water bath at atemperature of about 56° C. for about 1 hour.

Each of the above diluents was used to prepare three differentcalibrators containing different levels of human synthetic BNP(purchased from Peptide Institute (Osaka, Japan). Calibrator #A did notcontain any human synthetic BNP. Calibrator #B contained 50picograms/milliliter of human synthetic BNP. Calibrator #F contained2000 picograms/milliliter of human synthetic BNP. Each calibrator wasfiltered and placed in calibrator bottles and stored at 37° C. for up to14 days.

A BNP immunoassay was performed on an AxSYM® instrument (this instrumentis described in U.S. Pat. No. 5,358,691). In addition to the calibratorsprepared as described above, the following reagents were used in theassay:

1. Monoclonal antibody (MAb) 106.3 (capture antibody). This MAb binds toamino acids 5-13 on the BNP peptide. Monoclonal antibody 106.3 iscommercially available from Scios, Inc. (Sunnyvale, Calif.) and isdescribed in U.S. Pat. No. 6,376,207. A microparticle was coated withMAb 106.3 using the techniques described in U.S. Pat. No. 6,376,207, foruse in the assay. Specifically, MAb 106.3 was coated onto 0.2 μm-sizecarboxylate modified polystyrene particles (commercially available fromSeradyn, Inc., Indianapolis, In.) using EDAC coupling (EDAC is generallyused as a carboxyl activating agent for amide bonding with primaryamines. In addition, it reacts with phosphate groups. It is used inpeptide synthesis, crosslinking proteins to nucleic acids and inpreparing immunoconjugates. The chemical formula for EDAC is1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride. EDAC iscommercially available from Interchim (France)). Particles were washedand overcoated with BSA. After a final wash, particles were stored at2-8° C. in a buffer containing sucrose, sodium azide and BSA.

2. Monoclonal antibody BC203 (reporter antibody). This MAb binds toamino acids 27-32 on the BNP peptide. Monoclonal antibody BC203 isavailable from Shionogi, Inc. (Osaka, Japan). Monoclonal antibody BC203is conjugated to alkaline phosphatase. BC203 was conjugated to alkalinephosphatase by Axis Shield Diagnostics (Dundee, Scotland, U.K.) in atypical coupling procedure using Traut's Reagent (which is2-iminothiolane and is commercially available from Interchim (France))and SMCC (SMCC is a heterobifunctional cross-linker whose formula issuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate. SMCC iscommercially available from Interchim (France)). The stock conjugate wasstored in a buffer containing BSA, fish gelatin, Brij-35 (Brij-35 ispolyoxyethyleneglycol dodecyl ether and is a detergent that iscommercially available from Sigma-Aldrich, St. Louis, Mo.) and sodiumazide.

3. 4-methylumbelliferyl phosphate substrate (1.2 mM, in buffer,containing 0.1 sodium azide), which is commercially available fromAbbott Laboratories, Abbott Park, Ill.

4. The AxSYM® instrument system uses two (2) buffers: a Matrix Cell Washand a Line Diluent. The Matrix Cell Wash is Tris buffered salinecontaining sodium azide and anti-microbial agents and is used to washthe particle reaction mixture. The Line Diluent is Phosphate buffercontaining sodium azide and anti-microbial agents, and is used to rinsethe AxSYM® probes in between each aspiration. The Matrix Cell Wash andLine Diluent are commercially available from Abbott Laboratories.

The 0.2 μm microparticles coated with the capture antibody in a buffercontaining sucrose, sodium azide and BSA were pipetted by the samplingprobe into the appropriate wells of the reaction vessel in the samplingcenter. An aliquot containing one of the calibrators described above inTable 1 was delivered to the same well of the reaction vessel as themicroparticles to form a reaction mixture. The reaction vessel wastransferred to the processing center. The reaction mixture was incubatedfor approximately 12 minutes at a temperature of about 34° C. After theincubation, an aliquot of the reaction mixture was transferred to thematrix cell of the AxSYM® instrument. The reaction mixture was thenwashed with the Matrix Cell Wash at 1 pulse of 100 μL to remove any ofthe calibrator that was not captured.

MAb BC203-conjugate at about 0.75 μg/mL in a buffer containing BSA, fishgelatin, Brij-3 and sodium azide, was dispensed onto the matrix cell andthe resulting combination was incubated for approximately 12 minutes ata temperature of about 34° C. The matrix cell was washed with the MatrixCell Wash at 5 pulses of 50 μL to remove the unbound materials.

A solution of the substrate, 4-methylumbelliferyl phosphate (MUP) inaminomethyl phosphate buffer (1.2 mM) was added to the matrix cell andthe rate of formation of 4-methylumbelliferone was measured byfluorescence reflectance. The fluorescent product,4-methylumbelliferone, was measured by the microparticle enzymeimmunoassay (MEIA) optical assembly of the AXSYM® instrument.

The AxSYM® system measured the alkaline phosphatase conversion of MUP to4-methylumbelliferone (MU) by the rate of formation of the fluorescentproduct, MU. The aforementioned rates are typically measured in countsper second per second. The assay was conducted twice for eachcalibrator. The results in Table 2A below and FIGS. 1A-1P show the meanof the two assays.

TABLE 2A Calibrator stability at accelerated stability condition (37°C.) for 0–14 days Cals 0 2 5 7 9 12 14 Con- 9.8 12.8 12.7 13.2 13.8 12.712.5 A Con- 39.9 41.6 37.9 39.4 37.8 33.8 30.9 B Con- 1725.2 1564.91410.1 1460.0 1477.6 1200.0 1107.9 F  1A 9.8 10.6 11.4 11.8 12.8 11.712.5  1B 37.3 33.6 30.6 30.2 32.9 29.2 26.4  1F 1703.2 1370.2 1184.41256.6 1276.4 1192.1 1102.4  2A 8.9 11.1 11.1 11.1 11.8 11.2 11.8  2B41.6 39.4 36.2 37.4 38.1 35.8 31.9  2F 1865.9 1637.2 1398.4 1556.31562.7 1369.8 1309.2  3A 10.7 11.3 13.2 13.9 11.7 13.7 13.3  3B 34.439.9 33.6 37.9 36.0 34.0 31.7  3F 1746.1 1500.2 1325.2 1427.5 1315.01139.1 1199.9  4A 10.5 11.2 12.0 11.6 12.5 12.6 12.4  4B 32.8 39.6 33.936.2 36.9 32.5 32.7  4F 1611.8 1531.2 1236.9 1435.9 1418.2 1234.7 1190.8 5A 9.2 11.4 12.3 12.0 11.9 12.1 12.3  5B 37.4 38.7 34.8 40.1 38.1 36.132.3  5F 1671.1 1519.9 1285.2 1546.7 1444.3 1290.9 1246.3  6A 10.4 11.312.6 12.2 13.1 13.2 11.7  6B 38.7 38.1 37.1 38.1 39.0 33.9 32.1  6F1721.3 1570.1 1350.8 1548.5 1455.7 1285.7 1387.7  7A 11.4 13.0 13.7 13.013.5 15.3 13.9  7B 44.8 47.3 41.5 45.5 47.0 39.0 38.3  7F 2036.9 1783.41531.5 1767.9 1787.4 1541.1 1483.2  8A 10.3 12.2 12.8 14.4 13.9 14.613.4  8B 38.5 43.5 35.4 39.4 39.8 35.6 35.1  8F 1637.5 1605.5 1305.21449.6 1468.7 1294.0 1286.0  9A 10.7 11.7 12.6 14.1 14.7 12.6 13.0  9B43.8 55.9 47.8 50.1 54.1 49.0 45.2  9F 1807.9 2072.7 1707.4 1867.61998.2 1713.0 1634.9 10A 10.1 12.0 12.5 13.2 13.4 12.6 12.1 10B 48.755.8 49.7 51.9 59.2 48.5 46.5 10F 1794.0 2124.1 1928.3 1839.6 2142.11812.5 1648.3 11A 11.0 11.4 13.3 14.0 14.0 14.3 12.9 11B 39.0 49.0 42.843.5 49.2 43.4 42.2 11F 1606.1 1887.1 1593.5 1633.5 1836.5 1529.1 1637.912A 10.0 12.2 12.9 13.6 13.4 13.0 12.7 12B 44.4 58.0 46.4 49.4 53.7 48.448.6 12F 1784.8 1993.3 1759.2 1740.2 1839.2 1614.6 1629.7 13A 10.1 11.912.2 12.2 12.9 12.2 12.6 13B 49.2 64.1 58.2 58.7 62.1 54.0 54.4 13F1996.7 2283.9 2023.6 2088.8 2283.5 1983.7 2171.4 14A 10.6 12.4 12.5 12.113.3 12.0 13.1 14B 42.7 56.4 54.0 56.5 56.8 47.8 52.6 14F 1864.3 2029.91861.4 1932.5 2064.8 1832.9 2092.6 15A 11.7 13.6 13.2 14.0 16.4 15.513.0 15B 49.8 62.7 54.7 58.2 59.1 56.5 55.4 15F 2106.2 2259.1 1995.22032.4 2247.6 2062.1 2150.2 16A 12.0 12.8 12.7 13.5 16.3 14.8 15.0 16B44.8 57.0 50.3 52.4 60.5 48.4 54.1 16F 1801.6 1889.1 1889.4 1794.11977.2 1738.8 1930.6

Based upon the results shown about in Table 2A, the stability ofcalibrators 13, 14, 15 and 16 further examined at 21 days, 35 days, 49days, 77 days, 96 days and 187 days. The results are shown below inTable 2B. The results shown are the mean of two assays.

TABLE 2B 21 35 49 77 96 110 125 131 159 187 Con-A 13.7 14.8 14.9 Con-B27.5 26.6 24.1 Con-F 992.2 795.4 650.6 13A 13.4 13.8 13.4 14.8 20.6 13B46.8 48.6 50.0 45.4 46.6 13F 1853.7 1895.4 1803.3 1579.1 1484.3 14A 11.115.2 14.3 15.6 20.5 14B 42.8 49.2 44.7 40.9 42.7 14F 1712.0 1736.81694.1 1428.1 1291.5 15A #N/A #N/A #N/A #N/A #N/A #N/A #N/A #N/A #N/A18.0 15B #N/A #N/A #N/A #N/A #N/A #N/A #N/A #N/A #N/A 42.6 15F #N/A #N/A#N/A #N/A #N/A #N/A #N/A #N/A #N/A 1297.3 16A 12.8 16.0 15.1 16.6 20.9#N/A #N/A #N/A #N/A 18.7 16B 43.8 49.4 42.9 46.1 45.3 #N/A #N/A #N/A#N/A 34.8 16F 1775.0 1646.2 1673.6 1490.2 1350.2 #N/A #N/A #N/A #N/A1030.6

The data in Table 2A and FIGS. 1A-1P demonstrate that calibrators havinga pH in the range of from about 4.0 to about 6.5, particularly at pH5.6, exhibited enhanced accelerated stability compared to similarformulations with a pH of about 7.4. In calibrators 1-8 and theexperimental control diluent calibrator, the rates for the “F”calibrators, i.e., 1F, 2F, 3F, 4F, 5F, etc. (hereinafter referred to asthe “Cal-F” rates) fell much faster through 14 days of stability studythan the Cal-F rates in calibrators 9-16 which were similar formulationsbut at a different pH.

The enhanced accelerated stability can be quantified by comparing theaverage percentage change in F Calibrator rates at 37° C. for 14 daysfor the eight F Calibrators at approximately pH 7.4 (Calibrators1F-8F;mean F Calibrator rate decreased by 27.1%) versus the eight FCalibrators in similar formulations but at approximately pH 5.6(Calibrators 9F-16F; mean F Calibrator rate increased 0.9%). In eachpaired calibrator diluent (e.g., 1 and 9, 2 and 10, etc.) in which theformulation is identical except for pH, the F Calibrator rates droppedmore for those at approximately pH 7.4 than those at approximately pH5.6. (Accelerated stability studies are well known in the art in whichmaterials are stressed at a higher temperature than the intended storagecondition. In describing a European standard for stability testing of invitro diagnostic medical devices (EN 13640:2002), Armstrong describesincubation at an elevated temperature of 25° C., 37° C. or 40° C. asexamples of accelerated stability testing. The Food and DrugAdministration also recognizes accelerated stability studies, forexample, the Center for Drug Evaluation and Research, Manual of Policiesand Procedures, issued directive MAPP 5226.1. Reaffirmation ofexpiration dating period for abbreviated applications. In the backgroundsection of this directive they describe accelerated stability conditionsand data submittable for supporting stability (expiration dating) ofgeneric drugs.)

EXAMPLE 2 BNP Immunoassay

Bulk diluents (“Dil”) of the following formulations were prepared.

Experimental Control Diluent=4% BSA, 1% PEG, 0.1% Tween, 1% Dextran,0.1% Proclin® 300, MEIA2 diluent.

Dil1=2% BSA, 0.1% Proclin® 300, MEIA2 diluent

Dil2=Same as Dil1.

Dil3=2% BSA, 0.1% Proclin® 300, MEIA2 diluent and 0.1% Tween.

Dil4=Same as Dil3.

Dil5=2% BSA, 0.1% Proclin® 300, MEIA2 diluent and 10 mM DTPA

Dil6—Same as Dil5.

Dil7=2% BSA, 0.1% Proclin® 300, MEIA2 diluent, 0.1% Tween, 10 mM DTPA.

Dil8=Same as Dil7.

Dil9=Same as Dil1.

Dil10=Same as Dil2.

Dil11=Same as Dil3.

Dil12=Same as Dil4.

Dil13=Same as Dil5.

Dil14=Same as Dil6.

Dil15=Same as Dil7.

Dil16=Same as Dil8.

After each of the above diluents was prepared, the pH was determined.The pH of diluents 7-16 was adjusted. Specifically, for diluents thatcontained DTPA, NaOH was added (drop wise) to adjust the pH (to about5.6). For the diluents that contained DTPA, NaOH was used to adjust thepH (to about 5.6 or 7.4). The pH was not adjusted for diluents 1-4. Thefinal pH for each diluent is shown below in Table 3.

TABLE 3 Dil1 - pH 7.41 Dil2 - pH 7.41* Dil3 - pH 7.39 Dil4 - pH 7.39*Dil5 - pH 7.41 Dil6 - pH 7.41* Dil7 - pH 7.40 Dil8 - pH 7.40* Dil9 - pH5.66 Dil10 - pH 5.66* Dil11 - pH 5.64 Dil12 - pH 5.64* Dil13 - pH 5.66Dil14 - pH 5.66* Dil15 - pH 5.64 Dil16 - pH 5.64* The pH of theexperimental control diluent was 7.4. *After final adjustment of the pH,these diluents (and the control) were also subjected to heat.Specifically, these diluents were heated in a water bath at atemperature of about 56° C. for about 1 hour.

Each of the above diluents was used to prepare three differentcalibrators containing different levels of human synthetic BNP(purchased from Peptide Institute (Osaka, Japan). Calibrator #A did notcontain any human synthetic BNP. Calibrator #B contained 50picograms/milliliter of human synthetic BNP. Calibrator #F contained2000 picograms/milliliter of human synthetic BNP. Each calibrator wasfiltered and placed in calibrator bottles and stored at 2-8° C. for upto 271 days.

A BNP immunoassay was performed on an AxSYM® instrument as described inExample 1. The assay was conducted twice for each calibrator. Theresults in Table 4 below and FIGS. 2A-2P show the mean of two assays.

TABLE 4 Calibrator stability at 2–8° C. for up to 271 days Cals 0 2 5 79 12 14 243 271 Con-A 9.8 13.2 12.3 14.6 14.8 14.8 14.4 19.9 16.4 Con-B39.9 52.7 48.3 54.3 57.9 51.0 58.3 43.8 39.9 Con-F 1725.2 1988.8 1867.61949.6 2229.4 1901.8 2123.8 1199.9 1239.2  1A 9.8 12.7 11.5 12.8 12.911.9 11.8 20.7 15.3  1B 37.3 49.4 45.3 50.2 51.4 46.5 52.8 39.0 36.4  1F1703.2 1928.9 1887.0 2004.1 2183.3 1846.8 2196.8 1125.8 1181.3  2A 8.911.6 11.7 12.3 12.6 11.5 11.5  2B 41.6 52.4 50.0 56.6 63.8 53.8 57.7  2F1865.9 2255.0 2055.9 2170.1 2298.1 2186.9 2327.8  3A 10.7 12.1 12.4 13.014.6 15.6 14.4 21.7 17.2  3B 34.4 47.1 43.9 52.3 49.6 48.3 51.5 39.636.8  3F 1746.1 1940.5 1919.3 2103.5 2173.1 1879.1 2232.9 1021.9 1030.5 4A 10.5 11.8 12.5 13.7 14.8 15.3 14.0 #N/A 16.8  48 32.8 42.6 42.8 45.447.1 44.9 48.6 #N/A 39.6  4F 1611.8 1863.5 1792.4 1859.7 1973.7 1781.12026.9 #N/A 1190.4  5A 9.2 12.0 12.1 13.9 12.3 12.0 12.2 21.1 15.1  5B37.4 46.7 43.6 48.8 55.5 48.8 50.9 44.0 39.9  5F 1671.1 1989.7 1907.41976.4 2133.1 1910.4 2110.1 1230.4 1319.5  6A 10.4 12.8 11.6 13.3 12.712.5 13.7  6B 38.7 44.9 43.8 46.3 50.0 45.2 50.9  6F 1721.3 1710.01906.6 1845.7 1954.2 1791.3 2027.4  7A 11.4 13.3 15.2 16.4 14.1 15.014.6 24.1 19.2  7B 44.8 52.7 60.2 58.5 63.7 56.5 65.4 48.0 41.5  7F2036.9 2120.2 2150.2 2205.2 2340.9 2138.6 2405.4 1329.9 1386.0  8A 10.313.0 12.9 15.1 14.2 16.0 14.6  8B 38.5 43.2 46.6 49.6 51.9 49.0 55.3  8F1637.5 1700.3 1765.6 1728.0 1856.6 1703.4 1963.0  9A 10.7 13.5 12.9 13.512.6 11.6 12.3 21.5 16.3  9B 43.8 51.5 56.6 54.4 60.1 56.5 61.9 55.750.4  9F 1807.9 1855.8 1985.5 2042.5 2078.3 2012.2 2382.1 1586.5 1669.110A 10.1 11.8 12.1 12.3 12.9 12.1 11.3 10B 48.7 50.4 50.4 54.7 56.6 56.065.1 10F 1794.0 1922.5 2119.9 2051.4 2153.9 2130.1 2387.8 11A 11.0 12.512.1 13.4 14.9 14.5 14.6 22.5 16.9 11B 39.0 42.9 46.7 51.3 53.9 46.454.0 50.2 45.8 11F 1606.1 1606.4 1864.1 1942.5 1894.7 1740.9 1992.91361.7 1444.0 12A 10.0 12.3 13.0 14.9 14.6 14.3 14.5 12B 44.4 49.4 57.562.8 58.5 54.4 62.9 12F 1784.8 1844.2 1999.4 2206.1 2074.8 2072.6 2389.013A 10.1 11.8 12.8 11.1 12.0 12.4 11.0 13B 49.2 53.9 62.3 69.2 62.3 61.365.1 13F 1996.7 2037.5 2187.6 2382.9 2225.7 2148.3 2168.7 14A 10.6 11.513.1 13.8 12.3 12.9 12.5 14B 42.7 48.6 56.0 62.2 54.0 51.7 51.6 14F1864.3 1820.8 2135.9 2194.2 2090.4 2093.0 2000.9 15A 11.7 13.8 14.4 13.815.5 14.2 15.3 22.2 17.7 15B 49.8 54.5 63.3 68.2 61.8 61.9 57.2 55.953.2 15F 2106.2 2059.9 2354.6 2414.5 2191.5 2130.9 2209.0 1722.8 1805.316A 12.0 12.2 14.0 15.2 14.7 14.3 14.2 21.8 DEPLETED 16B 44.8 48.0 53.059.9 55.1 53.3 54.5 50.5 DEPLETED 16F 1801.6 1760.3 1943.0 2093.6 1907.91866.5 1861.8 #N/A DEPLETED

The data in Table 4 and FIGS. 2A-2P demonstrate that calibrators havinga pH in the range of from about 4.0 to about 6.5 (calibrators 9-16)exhibited less of a signal decrease than the corresponding calibrators1-8 at a pH of about 7.4. This enhanced long term stability was observedthrough 271 days at 2-8° C.

EXAMPLE 3 BNP Immunoassay

Bulk diluents (“Dil”) of the following formulations were prepared.

Dil1=2% BSA, 0.1% Proclin® 300, 10 mM DTPA

Dil2=2% BSA, 0.1% Proclin® 300, 10 mM DTPA, 100 mM NaCl, 0.1% sodiumazide in water

Dil3=2% BSA, 0.1% Proclin® 300, 10 mM DTPA, 100 mM NaCl, 0.1% sodiumazide in water

Dil4=2% BSA, 0.1% Proclin® 300, 10 mM DTPA, 100 mM NaCl, 0.1% sodiumazide in water

Dil5=2% BSA, 0.1% Proclin® 300, 10 mM DTPA, 100 mM NaCl, 0.1% sodiumazide in water

Dil6=2% BSA, 0.1% Proclin® 300, 10 mM DTPA, 100 mM NaCl, 0.1% sodiumazide in water

Dil7=2% BSA, 0.1% Proclin® 300, 10 mM DTPA, 100 mM NaCl, 0.1% sodiumazide in water

Dil8=2% BSA, 0.1% Proclin® 300, 10 mM DTPA, 100 mM NaCl, 0.1% sodiumazide in water

Dil9=2% BSA, 0.1% Proclin® 300, 10 mM DTPA, 100 mM NaCl, 0.1% sodiumazide in water

Dil10=2% BSA, 0.1% Proclin® 300, 10 mM DTPA, 100 mM NaCl, 0.1% sodiumazide in water

In addition, to the components listed above, diluent 1 also containedMEIA2 as a base diluent. Diluents 3-6 also contained a solution ofsodium acetate and a solution of acetic acid. Diluents 7-10 alsocontained a solution of monobasic sodium phosphate and a solution ofdibasic sodium phosphate.

After each of the above diluents was prepared, the pH was determined.The pH of diluents 3-10 was adjusted. Specifically, the pH of diluents3-10 was adjusted with NaOH. The pH was not adjusted for diluents 1-2.The final pH for each diluent is shown below in Table 5.

TABLE 5 Dil1 - pH 5.66* Dil2 - pH 3.91 Dil3 - pH 3.47 Dil4 - pH 4.50Dil5 - pH 5.02 Dil6 - pH 5.49* Dil7 - pH 6.10* Dil8 - pH 6.51* Dil9 - pH7.04* Dil10 - pH 10.04* *After final adjustment of the pH, thesediluents were also subjected to heat. Specifically, these diluents wereheated in a water bath at a temperature of about 60° C. for about 1hour.

Each of the above diluents was used to prepare three differentcalibrators containing different levels of human synthetic BNP(purchased from Peptide Institute (Osaka, Japan). Calibrator #A did notcontain any human synthetic BNP. Calibrator #B contained 50picograms/milliliter of human synthetic BNP. Calibrator #F contained2000 picograms/milliliter of human synthetic BNP. Each calibrator wasfiltered and placed in calibrator bottles and stored at 37° C. and 2-8°C. for up to 252 days.

A BNP immunoassay was performed on an AxSYM® instrument as described inExample 1, the only change being that about 0.50 μg/mL of MAbBC203-conjugate in a conjugate buffer containing BSA, fish gelatin,Brij-35 and sodium azide was used. The assay was conducted twice foreach calibrator. The results in Table 6 below and FIGS. 3A-3T show themean of the two assays.

TABLE 6 Calibrator stability at 37° C. or 2–8° C. for up to 252 days 0 37 14 21 28 35 56 77 37° C.  1A 14.0 12.3 12.8 12.4 11.5 13.8 14.5 18.916.7  1B 92.0 74.5 76.8 79.9 61.9 68.7 62.9 64.1 49.6  1F 2413.7 2318.62056.3 2200.7 2004.3 1943.7 1803.7 1524.1 1499.2  5A 11.5 12.7 14.4 17.911.4 16.0 15.2 17.6 17.2  5B 96.0 81.5 77.5 76.6 61.8 70.6 67.0 61.658.1  5F 2600.2 2406.2 2206.8 2212.0 2053.6 2078.9 2001.7 1784.2 1800.4 2A 11.9 28.0 104.2 49.7  2B 91.7 68.2 95.4 58.5  2F 2477.4 2080.1 54.7122.3  4A 11.3 35.6 27.7 55.8 #N/A #N/A #N/A #N/A #N/A  4B 87.1 74.682.4 77.2 #N/A #N/A #N/A #N/A #N/A  4F 1966.6 1769.2 1686.9 1901.0 #N/A#N/A #N/A #N/A #N/A  6A 12.6 15.6 14.5 13.8 12.3 15.0 15.0 18.2 16.1  6B86.3 117.7 149.2 67.4 53.7 61.3 59.2 53.8 43.9  6F 2548.6 2359.5 2187.12134.9 1997.1 1902.5 1857.1 1653.4 1553.2  7A 11.4 11.2 17.3 12.5 11.614.8 13.5 17.3 18.4  7B 99.0 71.3 70.5 68.4 56.5 55.6 50.5 46.8 40.5  7F2617.0 2308.4 2056.5 2120.1 1887.1 1780.3 1641.6 1326.0 1157.2  8A 11.411.3 17.6 16.2 10.3 12.6 #N/A #N/A #N/A  8B 92.1 65.9 60.5 55.9 44.043.7 #N/A #N/A #N/A  8F 2474.9 2073.8 1882.4 1754.6 1572.6 1385.9 #N/A#N/A #N/A  9A 12.5 10.4 13.9 14.3 13.3 13.8  9B 96.3 63.3 58.4 47.4 36.734.6  9F 2686.1 2175.3 1940.8 1643.5 1495.3 1158.3  3A 11.3 40.6 29.242.1  3B 25.7 24.9 20.7 30.7  3F 533.9 28.9 23.4 27.8 10A 15.1 12.4 15.413.9 10B 46.4 21.7 18.4 13.5 10F 1518.8 663.9 261.9 83.4 2–8° C.  1A14.0 11.3 17.8 15.7 12.9 13.4 13.4 16.9 16.6  1B 92.0 83.0 100.0 89.879.0 80.7 76.6 77.7 71.5  1F 2413.7 2435.9 2330.2 2333.4 2429.0 2396.12269.7 2086.2 2068.8  5A 11.5 10.6 13.9 12.2 11.4 14.0 13.1 18.5 19.2 5B 96.0 86.3 88.4 92.5 79.1 88.5 81.5 75.6 70.0  5F 2600.2 2480.72279.1 2548.5 2518.7 2415.9 2379.8 2210.6 2056.5  2A 11.9 10.7 17.4 14.5#N/A #N/A #N/A #N/A #N/A  2B 91.7 80.6 87.0 78.0 #N/A #N/A #N/A #N/A#N/A  2F 2477.4 2420.9 2341.0 2430.7 #N/A #N/A #N/A #N/A #N/A  4A 11.310.2 14.6 15.0 #N/A #N/A #N/A #N/A #N/A  4B 87.1 75.7 85.1 84.1 #N/A#N/A #N/A #N/A #N/A  4F 1966.6 1926.3 1788.1 2098.1 #N/A #N/A #N/A #N/A#N/A  6A 12.6 11.2 15.6 17.3 11.5 14.4 13.7 17.7 17.2  6B 86.3 69.2 78.280.1 72.6 73.3 66.1 68.4 56.9  6F 2548.6 2423.7 2307.8 2469.5 2529.92405.9 2236.6 2105.0 2032.9  7A 11.4 10.7 13.1 12.1 11.8 16.5 15.6 18.216.3  7B 99.0 84.7 89.3 89.6 87.0 87.6 83.8 82.9 73.0  7F 2617.0 2511.72478.8 2634.6 2619.5 2504.0 2370.4 2184.6 2157.2  8A 11.4 11.0 14.2 12.712.5 14.2 #N/A #N/A #N/A  8B 92.1 84.7 84.7 86.7 73.4 75.8 #N/A #N/A#N/A  8F 2474.9 2524.4 2286.1 2489.0 2354.1 2284.6 #N/A #N/A #N/A  9A12.5 10.3 14.0 13.0 12.5 15.1 #N/A #N/A #N/A  9B 96.3 87.1 95.5 91.284.0 86.3 #N/A #N/A #N/A  9F 2686.1 2682.7 2508.2 2662.3 2448.3 2372.0#N/A #N/A #N/A  3A 11.3 9.8 16.8 13.6  3B 25.7 28.6 33.9 29.6  3F 533.9560.8 602.5 545.7 10A 15.1 11.9 17.5 16.6 10B 46.4 37.3 41.2 37.4 10F1518.8 1407.2 1226.0 1238.1 91 106 112 140 168 193 224 252 37° C.  1A19.0 #N/A 25.3 31.8 36.5  1B 50.6 #N/A 47.7 51.4 60.2  1F 1248.8 #N/A1065.4 784.6 726.2  5A 17.5 #N/A 23.1 28.5 38.8  5B 55.4 #N/A 53.7 58.773.9  5F 1712.5 #N/A 1598.9 1375.2 1512.3  2A  2B  2F  4A #N/A #N/A #N/A#N/A #N/A 57.1  4B #N/A #N/A #N/A #N/A #N/A 53.2  4F #N/A #N/A #N/A #N/A#N/A 85.0  6A 17.9 #N/A 26.4 28.6 44.3 31.3  6B 45.9 #N/A 49.1 48.1 58.141.8  6F 1517.0 #N/A 1266.3 1167.2 1061.1 885.9  7A 17.2 #N/A 26.5 27.540.7 48.4  7B 37.7 #N/A 44.9 42.6 62.8 48.4  7F 1117.5 #N/A 901.0 759.3744.5 619.0  8A #N/A #N/A #N/A #N/A #N/A 31.3  8B #N/A #N/A #N/A #N/A#N/A 37.8  8F #N/A #N/A #N/A #N/A #N/A 362.5 2–8° C.  1A 17.3 17.2 27.6#N/A #N/A depleted  1B 77.9 65.3 79.3 84.8 #N/A depleted  1F 1971.22024.2 1828.9 1799.3 1940.6 depleted  5A 17.9 18.3 22.2 28.3 44.5depleted  5B 67.6 67.4 77.2 79.7 #N/A depleted  5F 2157.9 2217.0 1992.11872.0 #N/A depleted  2A #N/A #N/A #N/A #N/A #N/A #N/A 19.4 16.4  2B#N/A #N/A #N/A #N/A #N/A #N/A 47.0 46.3  2F #N/A #N/A #N/A #N/A #N/A#N/A 1586.4 1577.9  4A #N/A #N/A #N/A #N/A #N/A 28.0 16.8 17.8  4B #N/A#N/A #N/A #N/A #N/A 79.3 56.7 61.1  4F #N/A #N/A #N/A #N/A #N/A 1501.01380.2 1373.2  6A 19.7 #N/A 29.0 29.8 42.4 31.5 21.9 19.5  6B 60.1 #N/A65.8 70.6 81.0 77.1 54.0 50.3  6F 1966.8 #N/A 1871.1 1754.6 1816.01634.6 1608.2 1591.8  7A 18.5 #N/A 27.7 31.1 42.1 32.3 20.5 18.4  7B70.5 #N/A 76.3 74.7 97.4 79.1 52.8 51.9  7F 2016.7 #N/A 1898.1 1788.81940.9 1809.8 1579.4 1653.1  8A #N/A #N/A #N/A #N/A #N/A 31.4 18.0 16.5 8B #N/A #N/A #N/A #N/A #N/A 64.2 45.4 43.9  8F #N/A #N/A #N/A #N/A #N/A1617.1 1430.4 1389.9  9A #N/A #N/A #N/A #N/A #N/A #N/A 22.4 19.1  9B#N/A #N/A #N/A #N/A #N/A #N/A 42.3 42.7  9F #N/A #N/A #N/A #N/A #N/A#N/A 1478.5 1497.4

The data in Table 6 and FIGS. 3A-3T demonstrate that calibrators havinga pH in the range of from about 4.0 to about 6.5 (calibrators indiluents 1, 4-8) exhibited less of a signal decrease than thecalibrators in diluents 2-3 (at pH's of 3.91 and 3.47, note that #3 hadlow signal even at 0 time) and calibrators in diluents 9-10 (pH's 7.04and 10.04) at 37° C. accelerated stability. At 2-8° C., little change inF Cal rates was observed through 14 days except for diluent #3 which hadlow signal even at 0 time and for diluent #10 which decreased by 18.5%.

EXAMPLE 4 Stabilized Test Sample

Three (3) human plasma samples with levels of BNP greater than 2000pg/mL were obtained from patients with heart failure. 120 μL of eachspecimen was diluted with 480 μL of AxSYM® BNP Standard Calibrator A(0.32 M acetate buffer, 2% BSA (w/v), 10 mM DTPA, 0.09% sodium azide(w/v), 0.1 ProClin® 300 (w/v), pH 5.5) (“Calibrator A”). Another 120 μLof each specimen was obtained and was not diluted with Calibrator A(these samples are referred to as “undiluted plasma samples”).Immediately after dilution, the undiluted plasma samples (referred toherein as “neat”) and the diluted plasma samples were placed on theAxSYM® instrument system and analyzed for BNP using the AxSYM® BNPreagent kit (The AxSYM® BNP reagent kit contains the AxSYM® BNP ReagentPack (the AxSYM® BNP Reagent Pack contains 1 bottle (8.4 mL) Anti-BNP(Mouse Monoclonal) coated microparticles in Tris Buffer, 1 bottle (13.2mL) Anti-BNP (Mouse, Monoclonal) alkaline phosphatase conjugate in TRISbuffer with protein stabilizers, and 1 bottle (14.1 mL) wash buffercontaining detergent), Reaction Vessels and Matrix Cells). The neatsample underwent an auto-dilution (⅕) by the AxSYM® instrument and thediluted samples were tested directly. Following analysis, the remainingneat samples and diluted samples were stored at room temperature (22°C.) for four (4) hours. After this time period, all of the dilutedsamples were reanalyzed in the same manner as they were originallytested. The results from this testing are shown below in Table 7. Theresults demonstrate that the samples diluted with Calibrator A(described above) remain stable (97% recovery) following four (4) hourstorage at room temperature, whereas the neat samples, stored underthese same conditions, showed only 85% recovery.

TABLE 7 0 time 4 hrs Room Temp. Manual %0 %0 Manual %0 Auto dilutiontime Auto time dilution time dilu- in Auto dilu- Auto in Auto sampletion Cal A dilution tion dilution Cal A dilution 6BNP04 2908 3033 104%2676 92% 3015 104% BNP15 2673 2493 93% 2432 91% 2541 95% BNP27 3349 308392% 2452 73% 3119 93% Average 97% 85% 97% recovery =

EXAMPLE 5 Stabilized Diluted Sample

Nine (9) human plasma samples with levels of BNP greater than 400 pg/mL,including three of the samples from Example 4, were obtained frompatients with heart failure. 120 μL of each specimen was diluted with480 μL of AxSYM® BNP Standard Calibrator A (0.32 M acetate buffer, 2%BSA (w/v), 10 mM DTPA, 0.09% sodium azide (w/v), 0.1 ProClin® 300 (w/v),pH 5.5) (“Calibrator A”). Another 120 μL of each specimen was obtainedand was not diluted with Calibrator A (these samples are referred to as“undiluted plasma samples”). Immediately after dilution, all of theundiluted plasma samples (referred to herein as “neat”) and the dilutedplasma samples were placed on the AxSYM® instrument system and analyzedfor BNP using the AxSYM® BNP reagent kit (The AxSYM® BNP reagent kitcontains the AxSYM® BNP Reagent Pack (the AxSYM® BNP Reagent Packcontains 1 bottle (8.4 mL) Anti-BNP (Mouse Monoclonal) coatedmicroparticles in Tris Buffer, 1 bottle (13.2 mL) Anti-BNP (Mouse,Monoclonal) alkaline phosphatase conjugate in TRIS buffer with proteinstabilizers, and 1 bottle (14.1 mL) wash buffer containing detergent),Reaction Vessels and Matrix Cells). Following analysis, all of thediluted samples were stored at room temperature (22° C.). After four(4), five (5) and twenty-four (24) hours storage, the samples werereanalyzed in the same manner as they were originally tested. Theresults from this testing are shown below in Table 8. The dilutedconcentrations were multiplied by their dilution factors (5) and thendivided by the corresponding neat values to calculate dilution recovery.The results demonstrate that the samples diluted with Calibrator A(described above) remained stable following 4, 5 or 24 hour (102%, 97%,and 111% dilution recovery respectively) storage at room temperature.

TABLE 8 Diluted concentrations Dilution Recovery Sample Neat Value 0time 4 hours 5 hours 24 hours 0 time 4 hours 5 hours 24 hours A 2908 607603 — — 104% 104%  — — B 2673 499 508 — — 93% 95% — — C 3349 617 624 — —92% 93% — — D 1500 302 — 302 — 101% — 101% — E 2219 547 — 517 — 123% —116% — F 1680 300 — 327 — 89% —  97% — G 883 206 — 204 215 117% — 116%122% H 2160 456 — 479 541 106% — 111% 125% I 825 160 — 162 161 97% — 98%  98% J 494 96 — 101 100 97% — 102% 101% average = 102% 97% 106%111%

EXAMPLE 6 Comparison with Other Sample Diluents

Four (4) human plasma samples with BNP levels greater than 180 pg/mL,were obtained from patients with heart failure. 120 μL of each specimenwas diluted with 480 μL of AxSYM® BNP Standard Calibrator A (0.32 Macetate buffer, 2% BSA (w/v), 10 mM DTPA, 0.09% sodium azide (w/v), 0.1ProClin® 300 (w/v), pH 5.5) (“Calibrator 15 A”). Another 120 μL of eachspecimen was diluted with 480 μL of Multi-diluent I (Bayer HealthcareLLC, Tarrytown, N.Y.). Another 120 μL of each specimen was obtained andwas not diluted with Calibrator A or Multi-diluent I (these samples arereferred to as “undiluted plasma samples”). Immediately after dilution,all of the undiluted plasma samples (referred to herein as “neat”) andall of the diluted plasma 20 samples (both those diluted with CalibratorA and with Multi-diluent I) were placed on the AxSYM® instrument systemand analyzed for BNP using the AxSYM® BNP reagent kit (The AxSYM® BNPreagent kit contains the AxSYM® BNP Reagent Pack (the AxSYM® BNP ReagentPack contains 1 bottle (8.4 mL) Anti-BNP (Mouse Monoclonal) coatedmicroparticles in Tris Buffer, 1 bottle (13.2 mL) Anti-BNP (Mouse,Monoclonal) alkaline phosphatase conjugate in TRIS buffer with proteinstabilizers, and 1 bottle (14.1 mL) wash buffer containing detergent),Reaction Vessels and Matrix Cells). Following analysis, all of thediluted samples were stored at room temperature (22° C.). After five (5)hours and twenty-four (24) hours storage, the samples were reanalyzed inthe same manner as they were originally tested. The results from thistesting are shown below in Table 9. The diluted concentrations weremultiplied by their dilution factors (5) and then divided by thecorresponding neat values to calculate dilution recovery. The resultsdemonstrate that the samples diluted with Calibrator A (described above)remain stable following five (5) or twenty-four (24) hours (106% and112% recovery respectively) storage at room temperature with a dilutionrecovery of 110% of expected (range from 102% to 120%). The samplesdiluted in Multi-diluent I showed progressive decline over time withrecoveries at 5 and 24 hours of 67% and 54%. The 0 time dilutionrecovery was 121% (range from 84% to 216%).

TABLE 9 Diluted concentrations Neat 24 Dilution Recovery Sample Value 0time 5 hours hours 0 time 5 hours 24 hours Standard Calibrator A diluentA 2015 438 408 413 109% 101% 102% B 2069 448 455 505 108% 110% 122% C1005 205 208 — 102% 103% — D 183 44 40 — 120% 109% — Multi-diluent(Bayer) A 2015 359 280 201 89% 69%  50% B 2069 400 338 238 97% 82%  58%C 1005 168 137 — 84% 68% — D 183 79 18 — 216% 49% — Calibrator A average= 110% 106% 112% Multi-diluent average = 121% 67%  54%

1. A method of stabilizing a test sample for use in a ligand bindingassay for measuring a level of a natriuretic peptide in said testsample, wherein the method comprises the step of: mixing from about 5%to about 95% (v/v) of at least one diluent having a pH of from about 4.0to about 6.0 with at least one biological sample derived from serum,plasma, whole blood or other bodily fluids and that contains at leastone natriuretic peptide, to form a stabilized test sample having a pHfrom about 4.0 to about 6.5.
 2. The method of claim 1, wherein thenatriuretic peptide is a natural peptide selected from the groupconsisting of human natural atrial natriuretic peptide, human naturalB-type natriuretic peptide, human natural C-type natriuretic peptide andhuman natural Dendroaspis natriuretic peptide.
 3. The method of claim 1,wherein from about 70% to about 90% (v/v) of a diluent is mixed with atleast one biological sample.
 4. The method of claim 1, wherein thediluent has a pH of from about 5.4 to about 5.6.
 5. The method or claim1, wherein said diluent comprises at least one natriuretic stabilizingcompound.
 6. The method of claim 5, wherein said natriuretic stabilizingcompound is a protein or a polymer.
 7. The method of claim 6, whereinthe protein is selected from the group consisting of bovine serumalbumin, bovine gamma globulin, and a non-fat dry milk.
 8. The method ofclaim 6, wherein the polymer is selected from the group consisting ofpolyethylene glycol, dextran, dextran sulfate and polyvinyl pyrrolidone.9. The method of claim 1, wherein the diluent further comprises at leastone buffer, or at least one acid, or at least one base, or combinationsof at least one buffer, at least one acid and/or at least one base. 10.The method of claim 9, wherein said buffer is selected from the groupconsisting of an acetate buffer, a citrate buffer, a phosphate bufferand combinations thereof.
 11. The method of claim 9, wherein said acidis selected from the group consisting of acetic acid, citric acid,diethylenetriaminepentaacetic acid, hydrochloric acid and combinationsthereof.
 12. The method of claim 9, wherein said base is sodiumhydroxide.